Variants of vegfr and their use in the diagnosis and treatment of pregnancy associated medical conditions

ABSTRACT

An isolated polypeptide comprising an amino acid sequence at least 70% homologous to SEQ ID NO: 4 and an isolated polynucleotide encoding same are disclosed. A polynucleotide comprising a nucleic acid sequence capable of specifically hybridizing to the isolated polynucleotide and an isolated antibody comprising an antigen recognition domain which specifically binds the isolated polypeptide are also disclosed. Pharmaceutical compositions, methods of diagnosing and treating comprising same are also disclosed.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/448,404 filed on Jan. 13, 2010, which is a National Phase of PCTPatent Application No. PCT/IL2007/001589 filed on Dec. 20, 2007, whichclaims the benefit of priority under 35 USC §119(e) of U.S. ProvisionalPatent Application No. 60/875,822 filed on Dec. 20, 2006. The contentsof the above applications are all incorporated by reference as if fullyset forth herein in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to isolatedpolypeptides and polynucleotides encoding same for the diagnosis andtreatment of VEGF-associated medical conditions.

Vascular endothelial growth factor (VEGF), an endothelial specificmitogen, plays a key role in promoting both vasculogenesis andangiogenesis. VEGF plays an important regulatory function in theformation of new blood vessels during embryonic vasculogenesis and inangiogenesis during adult life.

The activities of VEGF are mediated primarily by its interaction withtwo high-affinity receptor tyrosine kinases: fms-like tyrosine kinase-1(Flt-1/VEGFR-1) and kinase-insert domain region (KDR/Flk-1/VEGFR-2) bothof which are expressed on vascular endothelial cell surfaces.Alternative splicing of Flt-1 results in the production of anendogenously secreted protein referred to as soluble Flt1 (sFlt1), whichlacks the cytoplasmic and transmembrane domains but retains theligand-binding domain [He et al., Mol. Endocrinol. (1999) 13: 537-545].Thus, sFlt1 can antagonize circulating VEGF by binding it and preventingthe interaction of VEGF with its endogenous receptors. sFlt1 also bindsand antagonizes placental growth factor (PlGF), another member of theVEGF family, which is produced predominantly in the placenta, as well asof another VEGF family member known as VEGF-B.

VEGF is an important mediator of angiogenesis in a number ofpathological conditions including tumor formation and metastasis ofsolid tumors. Numerous inhibitors of the VEGF/VEGF receptor pathway(e.g. monoclonal antibodies specific for VEGF) have been shown toprevent tumor growth via an antiangiogenic mechanism [Kim et al., Nature(1993) 362(6423):841-4].

Preeclampsia, the most common, dangerous, unpredictable complication ofpregnancy is a major cause of maternal, fetal, and neonatal mortalityworldwide. While the cause of preeclampsia remains unclear, theprinciple cause appears to be inadequate blood supply to the placentamaking it release hormones or chemical agents that cause maternalendothelial dysfunction, alterations in metabolism and inflammation[Drife J O, Magowan (eds) Clinical Obstetrics and Gynecology, chapter39, pp 367-370]. These consequently lead to hypertension associationwith proteinuria in the mother along with impaired placental blood flow,fetal growth restriction and consequential fetal oxidative stress.

During pregnancy, the major source of circulating sFlt1 is the placentaand only minor amounts are produced by other tissues (e.g. byendothelial cells and monocytes). Recent investigations have supportedthe finding that placental expression and serum levels of sFlt-1 areupregulated in preeclamptic pregnancies, in conjunction with decreasedlevels of circulating free VEGF and free PlGF, compared to normalpregnancies [Maynard et al., J. Clin. Invest. (2003) 111: 649-658]. Theincrease in sFlt1, followed by a decrease in free PlGF and free VEGF,was found to precede the onset of clinical disease by several weeks andappears to be more pronounced in severe and early onset preeclampsia[Levine et al., N. Engl. J. Med. (2004) 350: 672-683]. Postpartum,sFlt-1 levels decrease dramatically in both normal and preeclampticpregnancies [Maynard et al., supra]. Thus, excess sFlt1, by neutralizingVEGF and PlGF, may play a crucial role in the pathogenesis of thematernal syndrome in preeclampsia.

Lam et al. [Lam et al, Hypertension (2005) 46(5): 1077-85] review thepossibility of measuring circulating angiogenic proteins (e.g. PlGF) oranti-angiogenic proteins (e.g. sFlt-1) in the blood and urine ofpregnant women as a diagnostic and screening tool for predictingpreeclampsia. They have examined odds ratios, sensitivity andspecificity for various sFlt-1 cutoff values in different trimesters.Lam et al. describe a strong correlation between high sFlt-1 levels andthe risk and presence of preeclampsia. Furthermore, they have yieldedthe conclusion that the higher the sFlt-1 level, the more predictive itis of preeclampsia.

PCT Publication No. WO 2006/069373 discloses methods, compositions andkits for diagnosis of preeclampsia and hypertensive disorders inpregnancy. More specifically, WO 2006/069373 teaches assessment ofpreeclampsia or predisposition to preeclampsia by monitoring the levelsof angiogenic factors, specifically VEGF, PlGF and sFlt-1, in urinarysamples of pregnant women. WO 2006/069373 teaches that the higher thelevel of sFlt-1, the more predictive it is of preeclampsia. Furthermore,according to WO 2006/069373, preeclampsia is associated with asignificant decrease in PlGF and significant increase in VEGF urineconcentrations.

U.S. Publication No. 20050148040 discloses methods and compositions forscreening of gestational disorders (e.g., gestational diabetes,preeclampsia and gestational hypertension) using specific biomarkers.The biomarkers taught are insulin resistance biomarkers [e.g., sexhormone binding globulin (SHBG)] and angiogenesis biomarkers includingsFlt-1. More specifically, alterations in two pathways, insulinresistance (e.g., as evidenced by low serum levels of SHBG) andangiogenesis (e.g., as evidenced by low PlGF or high sFlt1), whencombined can be used to predict gestational disorders.

U.S. Publication No. 20050025762 discloses methods for diagnosing andtreating preeclampsia and eclampsia. U.S. Publication No. 20050025762teaches treating or preventing preeclampsia and eclampsia usingcompounds that increase VEGF or PlGF levels (e.g., nicotine, adenosine),using compounds that decrease sFlt-1 levels (e.g., purified sFlt-1antibody, an sFlt-1 antigen-binding fragment, small interfering RNAs, ordouble-stranded RNA) such as compounds that bind sFlt-1 and block growthfactor binding (e.g., chemical compound, polypeptide, peptide,antibody).

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided an isolated polypeptide comprising an amino acidsequence at least 70% homologous to SEQ ID NO: 4 as determined byprotein BLAST algorithm(http://wwwdotncbidotnlmdotnihdotgov/blast/Blastdotcgi).

According to some embodiments of the invention, the isolated polypeptideis capable of binding VEGF.

According to some embodiments of the invention, the isolated polypeptideis soluble.

According to some embodiments of the invention, the isolated polypeptideis as set forth in SEQ ID NO: 4.

According to some embodiments of the invention, the isolated polypeptideis as set forth in SEQ ID NO: 2.

According to some embodiments of the invention, the isolated polypeptidefurther comprises a heterologous amino acid sequence attached to theamino acid sequence.

According to some embodiments of the invention, the heterologous aminoacid sequence is selected from the group consisting of animmunoglobulin, a galactosidase, a glucuronidase, aglutathione-S-transferase (GST), a carboxy terminal peptide (CTP) fromchorionic gonadotrophin (CGβ), and a chloramphenicol acetyltransferase(CAT).

According to some embodiments of the invention, the isolated polypeptideis attached to a non-proteinaceous moiety.

According to some embodiments of the invention, the non-proteinaceousmoiety is selected from the group consisting of polyethylene glycol(PEG), Polyvinyl pyrrolidone (PVP), poly(styrene comaleic anhydride)(SMA), and divinyl ether and maleic anhydride copolymer (DIVEMA).

According to an aspect of some embodiments of the present inventionthere is provided an isolated polynucleotide comprising a nucleic acidsequence encoding the isolated polypeptide, wherein the isolatedpolynucleotide is not genomic Flt1.

According to some embodiments of the invention, the isolatedpolynucleotide is an mRNA or a cDNA.

According to an aspect of some embodiments of the present inventionthere is provided an isolated polynucleotide as set forth in SEQ ID NO:1 or 3.

According to an aspect of some embodiments of the present inventionthere is provided a nucleic acid construct comprising the nucleic acidsequence functionally attached to a cis-acting regulatory element.

According to an aspect of some embodiments of the present inventionthere is provided an isolated polynucleotide comprising a nucleic acidsequence capable of specifically hybridizing to the isolatedpolynucleotide and not to SEQ ID NO: 9.

According to an aspect of some embodiments of the present inventionthere is provided an isolated antibody comprising an antigen recognitiondomain which specifically binds the isolated polypeptide and not to SEQID NO: 10.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising the isolatedpolypeptide and a pharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising the isolatedpolynucleotide and a pharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising the antibodyand a pharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising the isolatedpolynucleotide and a pharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present inventionthere is provided a use of an agent capable of regulating an activityand/or expression of sFlt-14 (SEQ ID NO: 1 or 2) and not sFlt-1 (SEQ IDNO: 9 or 10), for the manufacture of a medicament identified fortreating a VEGF-associated medical condition.

According to some embodiments of the invention, the VEGF-associatedmedical condition is associated with reduced activity and/or expressionof VEGF and whereas the regulating comprises downregulating the sFlt-14.

According to some embodiments of the invention, the VEGF-associatedmedical condition is associated with excessive activity and/orexpression of VEGF and whereas the regulating comprises upregulating thesFlt-14.

According to some embodiments of the invention, the VEGF-associatedmedical condition is selected from the group consisting of preeclampsia,gestational diabetes, gestational hypertension, fetal growth restriction(FGR), fetal alcohol syndrome (FAS), cancer, corneal neovascularizationand hypertension.

According to some embodiments of the invention, the agent comprises theantibody.

According to some embodiments of the invention, the agent comprises theisolated polynucleotide.

According to some embodiments of the invention, the agent comprises theisolated polypeptide.

According to some embodiments of the invention, the agent comprises theisolated polynucleotide.

According to an aspect of some embodiments of the present inventionthere is provided a method of detecting sFlt-14 (SEQ ID NO: 2) in abiological sample, the method comprising: (a) contacting the biologicalsample with the antibody such that the sFlt-14 and the antibody form acomplex; and (b) measuring a presence or a level of the complex tothereby detect sFlt-14 in the biological sample.

According to an aspect of some embodiments of the present inventionthere is provided a method of detecting sFlt-14 (SEQ ID NO: 1) in abiological sample, the method comprising: (a) contacting the biologicalsample with the isolated polynucleotide so as to form a hybridizationcomplex; and (b) measuring a presence or a level of the complex tothereby detect sFlt-14 in the biological sample.

According to some embodiments of the invention, the measuring iseffected by a method selected from the group consisting of PCR, RealTime PCR, RT PCR, nucleic acid sequence-based amplification (NASBA),Northern blot and in situ hybridization.

According to an aspect of some embodiments of the present inventionthere is provided a method of diagnosing a pregnancy-associated medicalcondition associated with maternal or fetal stress in a subject in needthereof, the method comprising detecting expression level of sFlt-14(SEQ ID NO: 1 or 2) in a biological sample of the subject using an agentcapable of recognizing sFlt-14 (SEQ ID NO: 1 or 2) and not sFlt-1 (SEQID NO: 9 or 10), wherein an expression level of the sFlt-14 above apredetermined threshold is indicative of the pregnancy-associatedmedical condition associated with maternal or fetal stress.

According to an aspect of some embodiments of the present inventionthere is provided a method of diagnosing a pregnancy-associated medicalcondition associated with maternal or fetal stress in a subject in needthereof, the method comprising detecting expression level of sFlt-14(SEQ ID NO: 1 or 2) in a biological sample, wherein the biologicalsample is of a gestation week 13 and on, and wherein an expression levelof the sFlt-14 above a predetermined threshold is indicative of thepregnancy-associated medical condition associated with maternal or fetalstress.

According to some embodiments of the invention, the condition isselected from the group consisting of preeclampsia, gestationaldiabetes, gestational hypertension, fetal growth restriction (FGR), andfetal alcohol syndrome (FAS).

According to some embodiments of the invention, the biological sample isselected from the group consisting of a urine sample, a blood sample, aserum sample, a placenta biopsy, a chorionic villus sample, and anamniotic fluid sample.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a schematic representation of the FLT1-full transmembranereceptor (top), the well-known soluble sFlt1 variant (middle), and thenovel sFlt-14 variant of the present invention (bottom). Arrows mark thebeginning of translation (ATG) and the stop (STOP) points. Exons areindicated by the dark numbered boxes and introns 13 and 14 are indicatedby the light numbered boxes.

FIG. 2A depicts the unique cDNA sequence of the novel sFlt-14 isoform.The sequence was cloned by 3′ RACE from a human preeclamptic placenta.The shown sequence starts near the beginning of exon 14 and ends with apoly A tail. The italic letters represent the coding region that isderived from exon 14 and intron 14. The stop codon is in bold. Theunderlined sequence is an Alu repeat nested in the 3′ UTR.

FIG. 2B depicts a window taken from the UCSC genome browser after ablast search with the sequence that appears above (FIG. 2A). The searchlocated three ESTs with a similar splicing pattern to the one found inthe above sequence: AI188382, N47911 and AA035437.

FIG. 3A depicts the amino acid sequence of sFlt-14 as deduced from itsmRNA. The original reading frame was kept, starting with the knowntranslation start point of sFlt-1 and transmembrane Flt. The amino acidsshared with the full transmembrane receptor (but not with sFlt-1) areunderlined. The unique 28 amino acids found only in the variant sFlt-14of the invention (from intron 14) are depicted in bold.

FIG. 3B depicts sequences of peptides synthesized in order to createspecific polyclonal antibodies which distinguish between sFlt-1 andsFlt-14. Of note, whereas CESS is unique to sFlt-14, CHFK candistinguish between the alternatively-spliced isoforms only inconjunction with analysis of the protein size.

FIGS. 4A-B are pictures depicting the relative abundance of the fullmembrane receptor Flt, sFlt-1 (also designated sFlt-13) and the novelsFlt-14 in different cell types. FIG. 4A shows the expression of thefull receptor Flt and its two alternatively-spliced variants sFlt-1 andsFlt-14 in endothelial cells (left column), normal placentae (middlecolumn) and preeclampsia placentae (right column). RNA blots werehybridized with a probe detecting an extracellular sequence common toall three (each yields a band of equal intensity, irrespective of size).Of note, endothelial cells preferentially produce the full receptor andsFlt1, whereas placentae predominantly produce sFlt-14. However, adramatic upregulation of sFlt-14 is exhibited in preeclampsia placentae;FIG. 4B shows the expression of sFlt-1 and sFlt-14 in Primary culturesof endothelial cells (EC) and in vascular smooth muscle cells (VSMC)isolated from a human saphena vein. Of note, sFlt1 and sFlt-14 areexclusively expressed by ECs and VSMCs, respectively. The bottom panelsof FIGS. 4A-B represent 28S and 18S RNA expression that was used as aloading control.

FIGS. 5A-D are pictures depicting sFlt-14 mRNA and protein expression inthe context of the preeclamptic placenta. FIGS. 5A-B showimmunohistochemical detection of the sFlt-14 protein using the specificCESS antibody; FIGS. 5C-D show in-situ hybridization with asFlt-14-specific probe (derived from intron 14) identifyingsFlt-14-expressing cells. Of note, massive expression of sFlt-14 mRNAand protein in syncytial knots of the preeclamptic placenta.

FIG. 6 is a western immunoblot image depicting immunoprecipitation ofthe sFlt-14 protein in normal term placentae with a CESS antibody orwith a Flt1 antibody.

FIG. 7 shows a mass-spectrometry identification of sFlt-14 (SEQ ID NOs:16-19).

FIG. 8 is a western immunoblot image depicting expression levels ofsFlt-14 proteins in serum and placentae samples of preeclampticsubjects. Protein detection was carried out using a specific sFlt-14antibody (CESS, directed against SEQ ID NO: 5).

FIGS. 9A-B depict characterization of sFlt proteins during the course ofpregnancy. FIG. 9A illustrates RNA expression of sFlt-1 and sFlt-14during different time points of normal gestation; and FIG. 9B showsquantification of the ratio of the two sFlt1 isoforms (sFlt-1 andsFlt-14) during different time points of normal gestation: weeks 9-11,week 13, and week 39.

FIGS. 10A-B depict sFlt-14 as a VEGF receptor. FIG. 10A showsrecombinant sFlt-14 and sFlt-1 proteins from the cellular fraction (c)or from the media (m). sFlt-14 is located at 115 Kd and 130 Kd, in thecellular fraction and media, respectively. sFlt-1 is located at 100 Kdand 120 Kd, in the cellular fraction and media, respectively; and FIG.10B shows a VEGF inhibition assay where VEGF was pre-incubated withsFlt-1 or sFlt-14 prior to addition of VEGF-R2 (by addition of growthmedium of Porcine Aortic Endothelial cells). VEGF-R2 phosphorylationlevels were measured as a function of added sFlt-14/VEGF ratio orsFlt-1/VEGF ratio. Of note, nearly complete inhibition of VEGF-R2phosphorylation was evident already at a 1:1 sFlt-14/VEGF ratio.

FIGS. 11A-B depict sFlt-14 expression in human cornea sections. FIG. 11Ashows immunohistochemistry of sFlt-14 using a specific antibody (CESSdirected against SEQ ID NO: 5); and FIG. 11A shows controlimmunohistochemistry using a pre immuned serum. Of note, sFlt-14 wasreadily seen in the corneal epithelia.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to isolatedpolypeptides and polynucleotides encoding same for the diagnosis andtreatment of VEGF-associated medical conditions.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

The soluble VEGF receptor sFlt-1, which specifically binds andantagonizes circulating VEGF and PlGF, has been previously contemplatedas the leading cause of preeclampsia and as such was suggested as amarker of and target for treating this condition [Maynard et al., supra;Levine et al., supra]. These findings were based on the use of clinicaltools such as antibodies and oligonucleotides directed to sequencesshared by the soluble and non-soluble VEGF receptors.

While reducing some embodiments of the present invention to practice,the present inventors have identified a novel VEGF receptor variant.This variant is soluble, secreted, comprises a unique amino acidsequence (SEQ ID NO: 4) and is expressed during preeclampsia. Usingantibodies or oligonucleotides specifically directed to the uniquesequence (SEQ ID NOs 4 or 3) of sFlt-14, the present inventors were ableto show that it is the sFlt-14 rather than sFlt-1 (supra) that is highlyexpressed in preeclampsia. These results prove beyond any doubt theclinical value of sFlt-14.

As is illustrated in the Examples section which follows, the novelsFlt-14 (SEQ ID NOs: 1 and 2) of the present invention differs from thefull transmembrane receptor Flt1 and from the known sFlt1 by comprisinga unique 28 amino acid sequence (SEQ ID NO: 4, see Example 1 and FIG. 1)derived by readthrough of intron 14. This novel sFlt-14 receptor isexpressed in placentae and is highly upregulated in preeclampticplacentae (see Example 3 and FIG. 4A). Moreover, the results presentedherein illustrate that trophoblastic cells within the syncytial knotsproduce sFlt-14 (Example 4 and FIGS. 5A-D). Thus, sFlt-14 provides avaluable indicator of preeclampsia or predisposition thereof.Furthermore, since sFlt-14 functions in antagonizing VEGFR ligands(e.g., VEGF), modulating sFlt-14 levels (e.g. downregulating orupregulating) may serve as a powerful tool in treatment of VEGFassociated conditions (hyper angiogenesis e.g., cancer andneovascularized cornea).

Thus, according to one aspect of the present invention there is providedan isolated polynucleotide comprising a nucleic acid sequence encoding apolypeptide comprising an amino acid sequence at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 87%, at least about 89%, at least about 90%, at least about91%, at least about 93%, at least about 95% or more say 100% identicalor homologous to SEQ ID NO: 4, wherein the isolated polynucleotide isnot genomic Flt1.

As used herein the phrase “an isolated polynucleotide” refers to asingle or double stranded nucleic acid sequences which is isolated andprovided in the form of an RNA sequence, a complementary polynucleotidesequence (Cdna) and/or a composite polynucleotide sequences (e.g., acombination of the above).

As used herein the phrase “complementary polynucleotide sequence” refersto a sequence, which results from reverse transcription of messenger RNAusing a reverse transcriptase or any other RNA dependent DNA polymerase.Such a sequence can be subsequently amplified in vivo or in vitro usinga DNA dependent DNA polymerase.

As used herein the phrase “composite polynucleotide sequence” refers toa sequence, which is at least partially complementary and partiallygenomic. A composite sequence can include some exonal sequences requiredto encode the polypeptide of the present invention, as well as someintronic sequences interposing therebetween. The intronic sequences canbe of any source, including of other genes, and typically will includeconserved splicing signal sequences. Such intronic sequences may furtherinclude cis acting expression regulatory elements (further explained indetail hereinbelow).

According to an exemplary embodiment of this aspect of the presentinvention the isolated polypeptide encoded by the polynucleotidedescribed herein is capable of binding a VEGFR ligand. Examples of suchligands include, without limitation, VEGF (VEGF-A, GeneBank AccessionNo. NP_(—)001020537), VEGF-B (GeneBank Accession No. NP_(—)003368) andPlacenta growth factor (PlGF, GeneBank Accession No. NP_(—)002623).

According to an exemplary embodiment, binding of the polypeptide isexpected to be in a range of about 10⁻⁹ M-10⁻¹² M.

According to an exemplary embodiment of this aspect of the presentinvention, the isolated polynucleotide is as set forth in SEQ ID NO: 1.Of note, it is suggested that naturally occurring forms of thepolynucleotide sequences of some embodiments of the present inventionare splice variants of the genomic Flt1. Examples of genomic Flt1 aredepicted in GeneBank Accession No. NC_(—)000013.9 region: complement(27773790 to 27967232) GI:51511729 for human genomic Flt1 and GeneBankAccession No. NC_(—)006480.2 region: complement (27975879 to 28168596)GI:114795054 for chimpanzee genomic Flt1 (see FIG. 1 showing exon/intronorganization).

The phrase “splice variant”, as used herein, refers to alternative formsof RNA transcribed from a VEGF receptor gene. Splice variation arisesnaturally through use of alternative splicing sites within a transcribedRNA molecule, or less commonly between separately transcribed RNAmolecules, and may result in several different mRNAs transcribed fromthe same gene. Splice variants may encode polypeptides having alteredamino acid sequence due to intron inclusion, exon exclusion or acombination of both. The term splice variant is also used herein todenote a polypeptide encoded by a splice variant of an mRNA transcribedfrom a gene.

According to an alternative embodiment the isolated polynucleotide ofthe present invention is as set forth in SEQ ID NO: 3.

It will be appreciated that homologues of the sequences describedhereinabove are also envisaged by the present invention. Accordingly,the polynucleotide of this aspect of the present invention may have anucleic acid sequence at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 87%, at leastabout 89%, at least about 90%, at least about 91%, at least about 93%,at least about 95% or more say 100% identical or homologous to SEQ IDNO: 1 or 3, as determined using BlastN software of the National Centerof Biotechnology Information (NCBI) using default parameters.

Thus, the present invention encompasses nucleic acid sequences describedhereinabove; fragments thereof, sequences hybridizable therewith,sequences homologous thereto, sequences encoding similar polypeptideswith different codon usage, altered sequences characterized bymutations, such as deletion, insertion or substitution of one or morenucleotides, either naturally occurring or man induced, either randomlyor in a targeted fashion.

Since the polynucleotide sequences of the present invention encodepreviously unidentified polypeptides, the present invention alsoencompasses novel polypeptides or portions thereof, which are encoded bythe isolated polynucleotides and respective nucleic acid fragmentsthereof described hereinabove.

Thus, according to another aspect of the present invention there isprovided an isolated polypeptide comprising an amino acid sequence atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 87%, at least about 89%, at least about90% at least about 91%, at least about 93%, at least about 95% or moresay 100% homologous to SEQ ID NO: 4 as determined by protein BLASTalgorithm (http://wwwdotncbidotnlmdotnihdotgov/blast/Blastdotcgi).

In an exemplary embodiment the isolated polypeptide is as set forth inSEQ ID NO: 2 or 4.

The present invention also encompasses fragments (e.g., as short as aspecific antigenic determinant e.g., at least about 6, at least about 8,at least about 10 at least about 20 amino acids such as derived from SEQID NO: 4) of the above described polypeptides and polypeptides havingmutations, such as deletions, insertions or substitutions of one or moreamino acids, either naturally occurring or man induced, either randomlyor in a targeted fashion. These fragments may be used to elicit antibodyproduction against the isolated polypeptides of the invention.

As used herein the phrase “an isolated polypeptide” refers to isolated,native peptides (either degradation products, synthetically synthesizedpeptides, or recombinant peptides), peptidomimetics (typically,synthetically synthesized peptides), and the peptide analogues peptoidsand semipeptoids, and may have, for example, modifications rendering thepeptides more stable while in a body or more capable of penetrating intocells. Such modifications include, but are not limited to: N-terminusmodifications; C-terminus modifications; peptide bond modifications,including but not limited to CH₂—NH, CH₂—S, CH₂—S═O, O═C—NH, CH₂—O,CH₂—CH₂, S═C—NH, CH═CH, and CF═CH; backbone modifications; and residuemodifications. Methods for preparing peptidomimetic compounds are wellknown in the art and are specified, for example, in Ramsden, C. A., ed.(1992), Quantitative Drug Design, Chapter 17.2, F. Choplin PergamonPress, which is incorporated by reference as if fully set forth herein.Further details in this respect are provided hereinbelow.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH3)-CO—); ester bonds(—C(R)H—C—O—O—C(R)—N—); ketomethylene bonds (—CO—CH2-); α-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl group, e.g., methyl; carba bonds(—CH2-NH—); hydroxyethylene bonds (—CH(OH)—CH2-); thioamide bonds(—CS—NH—); olefinic double bonds (—CH═CH—); retro amide bonds (—NH—CO—);and peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom. These modifications canoccur at any of the bonds along the peptide chain and even at several(2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr, and Phe, may be substituted forsynthetic non-natural acids such as, for instance,tetrahydroisoquinoline-3-carboxylic acid (TIC), naphthylelanine (Nol),ring-methylated derivatives of Phe, halogenated derivatives of Phe, ando-methyl-Tyr.

In addition to the above, the polypeptides of the present invention mayalso include one or more modified amino acids or one or more non-aminoacid monomers (e.g., fatty acids, complex carbohydrates, etc.).

The term “amino acid” or “amino acids” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine, and phosphothreonine; and other less common amino acids,including but not limited to 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine, and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids.

Thus, polypeptides of the present invention can be of a short lengthtypically 5-10, 10-20, 20-50, 50-100 amino acids in length and longere.g., 100-200, 200-300, 300-400, 400-500, 500-600, 600-733 amino acidsin length.

Mimetic technology may be used to generate peptides which are engineeredto have at least one modified feature as compared to the naturallyoccurring polypeptide (e.g., SEQ ID NO: 2) while maintaining abiological activity of interest e.g., VEGF binding, antibody binding andthe like.

Generation of peptide mimetics can be effected using various approacheswhich are well known in the art, including, for example, displaytechniques.

Thus, the present invention contemplates a display library comprising aplurality of display vehicles (such as phages, viruses or bacteria) eachdisplaying at least 5, at least 7, at least 11, at least 15, at least20, at least 25 consecutive amino acids derived from the isolatedpolypeptide sequence of sFlt-14 (e.g., SEQ ID NO: 2 and SEQ ID NO: 4).

Peptide mimetics can also be uncovered using computational biology.

According to an embodiment of the present invention, the isolatedpolypeptides are soluble.

As used herein the term “soluble” refers to the ability of the moleculesof the present invention to dissolve in a physiological aqueous solution(pH about 7, e.g., solubility level in aqueous media of >100 μg/ml)without substantial aggregation. Thus, it is readily understood thatsoluble sFlt-14 are preferably devoid of hydrophobic transmembranedomains.

Being soluble, the polypeptides of the present invention may besecreted. As depicted in the Example section which follows, the presentinventors have revealed, for the first time, that sFlt-14 (and notsFlt-1) is the soluble receptor found in the serum of preeclampticsubjects (Example 7 and FIG. 8). Thus, sFlt-14 is the major VEGFreceptor in the circulation of preeclamptic subjects.

The peptides of the present invention are preferably utilized in alinear form, although it will be appreciated that in cases wherecyclization does not severely interfere with peptide characteristics,cyclic forms of the peptide can also be utilized.

The peptides of the present invention may be synthesized by anytechniques that are known to those skilled in the art of peptidesynthesis. For solid phase peptide synthesis, a summary of the manytechniques may be found in: Stewart, J. M. and Young, J. D. (1963),“Solid Phase Peptide Synthesis,” W. H. Freeman Co. (San Francisco); andMeienhofer, J (1973). “Hormonal Proteins and Peptides,” vol. 2, p. 46,Academic Press (New York). For a review of classical solution synthesis,see Schroder, G. and Lupke, K. (1965). The Peptides, vol. 1, AcademicPress (New York).

In general, peptide synthesis methods comprise the sequential additionof one or more amino acids or suitably protected amino acids to agrowing peptide chain. Normally, either the amino or the carboxyl groupof the first amino acid is protected by a suitable protecting group. Theprotected or derivatized amino acid can then either be attached to aninert solid support or utilized in solution by adding the next aminoacid in the sequence having the complimentary (amino or carboxyl) groupsuitably protected, under conditions suitable for forming the amidelinkage. The protecting group is then removed from this newly addedamino acid residue and the next amino acid (suitably protected) is thenadded, and so forth; traditionally this process is accompanied by washsteps as well. After all of the desired amino acids have been linked inthe proper sequence, any remaining protecting groups (and any solidsupport) are removed sequentially or concurrently, to afford the finalpeptide compound. By simple modification of this general procedure, itis possible to add more than one amino acid at a time to a growingchain, for example, by coupling (under conditions which do not racemizechiral centers) a protected tripeptide with a properly protecteddipeptide to form, after deprotection, a pentapeptide, and so forth.

Further description of peptide synthesis is disclosed in U.S. Pat. No.6,472,505. A preferred method of preparing the peptide compounds of thepresent invention involves solid-phase peptide synthesis, utilizing asolid support. Large-scale peptide synthesis is described by AnderssonBiopolymers 2000, 55(3), 227-50.

In cases where large amounts of the peptides of the present inventionare desired, the polypeptides of the present invention can be generatedusing recombinant techniques such as described by Bitter et al. (1987)Methods in Enzymol. 153:516-544; Studier et al. (1990) Methods inEnzymol. 185:60-89; Brisson et al. (1984) Nature 310:511-514; Takamatsuet al. (1987) EMBO J. 6:307-311; Coruzzi et al. (1984) EMBO J.3:1671-1680; Brogli et al. (1984) Science 224:838-843; Gurley et al.(1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988,Methods for Plant Molecular Biology, Academic Press, NY, Section VIII,pp 421-463.

Briefly, an expression construct (i.e., expression vector), whichincludes the isolated polynucleotide of the present invention (e.g., SEQID NO: 1, 3), optionally in frame fused to a nucleic acid sequenceencoding a heterologous amino acid sequence (e.g. immunoglobulinsequence, as further described hereinbelow), positioned under thetranscriptional control of a regulatory element, such as a promoter (asexplained in detail hereinbelow), is introduced into host cells.

For expression in mammalian cells, pRK5-based vectors [Schall et al.,Cell, 61:361-370 (1990)]; and CDM8-based vectors [Seed, Nature, 329:840(1989)] can be used.

Methods of introducing the expression construct into a host cell arewell known in the art and include electroporation, lipofection andchemical transformation (e.g., calcium phosphate).

The “transformed” cells are cultured under suitable conditions, whichallow the expression of the polypeptide encoded by the nucleic acidsequence.

Following a predetermined time period, the expressed chimeric moleculeis recovered from the cell or cell culture, and purification is effectedaccording to the end use of the recombinant polypeptide.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation elements including constitutiveand inducible promoters, transcription enhancer elements, transcriptionterminators, and the like, can be used in the expression vector [see,e.g., Bitter et al., (1987) Methods in Enzymol. 153:516-544].

Other than containing the necessary elements for the transcription andtranslation of the inserted coding sequence (encoding the chimera), theexpression construct of the present invention can also include sequencesengineered to optimize stability, production, purification, yield ortoxicity of the expressed fusion protein.

A variety of prokaryotic or eukaryotic cells can be used ashost-expression systems to express the fusion protein coding sequence.These include, but are not limited to, microorganisms, such as bacteriatransformed with a recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vector containing the chimera coding sequence; yeasttransformed with recombinant yeast expression vectors containing thechimera coding sequence; plant cell systems infected with recombinantvirus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobaccomosaic virus, TMV) or transformed with recombinant plasmid expressionvectors, such as Ti plasmid, containing the chimera coding sequence.Mammalian expression systems are preferably used to express the chimeraof the present invention.

The choice of host cell line for the expression of the molecules dependsmainly on the expression vector. Eukaryotic expression systems arepreferred (e.g., mammalian and insects) since they allow posttranslational modifications (e.g., glycosylation). Another considerationis the amount of protein that is required. Milligram quantities oftencan be produced by transient transfections. For example, the adenovirusEIA-transformed 293 human embryonic kidney cell line can be transfectedtransiently with pRK5-based vectors by a modification of the calciumphosphate method to allow efficient expression. CDM8-based vectors canbe used to transfect COS cells by the DEAE-dextran method (Aruffo etal., Cell, 61:1303-1313 (1990); Zettmeissl et al., DNA Cell Biol. US,9:347-353 (1990)]. If larger amounts of protein are desired, themolecules can be expressed after stable transfection of a host cellline. It will be appreciated that the presence of a hydrophobic leadersequence at the N-terminus of the molecule will ensure processing andsecretion of the molecule by the transfected cells.

It will be appreciated that the use of bacterial or yeast host systemsmay be preferable to reduce cost of production. However since bacterialhost systems are devoid of protein glycosylation mechanisms, a postproduction glycosylation may be needed.

In any case, transformed cells are cultured under effective conditions,which allow for the expression of high amounts of recombinantpolypeptide. Effective culture conditions include, but are not limitedto, effective media, bioreactor, temperature, pH and oxygen conditionsthat permit protein production. An effective medium refers to any mediumin which a cell is cultured to produce the recombinant chimera moleculeof the present invention. Such a medium typically includes an aqueoussolution having assimilable carbon, nitrogen and phosphate sources, andappropriate salts, minerals, metals and other nutrients, such asvitamins. Cells of the present invention can be cultured in conventionalfermentation bioreactors, shake flasks, test tubes, microtiter dishes,and petri plates. Culturing can be carried out at a temperature, pH andoxygen content appropriate for a recombinant cell. Such culturingconditions are within the expertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, resultantproteins of the present invention may either remain within therecombinant cell, secreted into the fermentation medium, secreted into aspace between two cellular membranes, such as the periplasmic space inE. coli; or retained on the outer surface of a cell or viral membrane.

Following a predetermined time in culture, recovery of the recombinantprotein is effected.

Molecules of the present invention are preferably retrieved in“substantially pure” form. As used herein, “substantially pure” refersto a purity that allows for the effective use of the protein in theapplications, described hereinbelow.

As mentioned, the isolated polypeptide of this aspect of the presentinvention may further comprise a heterologous amino acid sequence.

As used herein the phrase “heterologous amino acid sequence” refers toan amino acid sequence which does not form a part of a naturallyoccurring sFlt-14 (e.g., SEQ ID NO: 2) amino acid sequence. Thissequence preferably confers solubility to the molecule of thisembodiment of the present invention, and preferably increases thehalf-life of the chimeric molecule in the serum.

The heterologous amino acid sequence is generally localized at theamino- or carboxyl-terminus of the isolated polypeptide of the presentinvention.

One or more heterologous amino acid sequences can be conjugated to thesFlt-14 amino acid sequence of the present invention. Examples ofheterologous amino acid sequences commonly used in fusion proteinconstruction include, but are not limited to, immunoglobulin,galactosidase, glucuronidase, glutathione-S-transferase (GST), carboxyterminal peptide (CTP) from chorionic gonadotrophin (CGβ) andchloramphenicol acetyltransferase (CAT).

The exact site at which fusion (conjugation) between the heterologoussequence and the sFlt-14 amino acid sequence is not critical and theoptimal site can be determined by routine experimentation as long asfunctionality of the polypeptide is maintained (e.g., VEGF binding).Methods of ligand binding assessment are well known in the art (e.g.,using a radiolabeled ligand in a binding assay, or an ELISA).

Additionally or alternatively as mentioned hereinabove the isolatedpolypeptide of the present invention may be attached to anon-proteinaceous moiety.

Thus, embodiments of the present invention provide an isolatedpolypeptide or polynucleotide being attached to a non-proteinaceousmoiety.

Such a conjugate molecule is highly stable (resistant to in-vivoproteolytic activity probably due to steric hindrance conferred by thenon-proteinaceous moiety) and may be produced using common solid phasesynthesis methods which are inexpensive and highly efficient, as furtherdescribed hereinbelow. However, it will be appreciated that recombinanttechniques may still be used, whereby the recombinant peptide product issubjected to in-vitro modification (e.g., PEGylation).

The phrase “non-proteinaceous moiety” as used herein refers to amolecule not including peptide bonded amino acids that is attached tothe above-described sFlt-14 amino acid sequence. According to anembodiment the non-proteinaceous moiety of this aspect of the presentinvention is a polymer or a co-polymer (synthetic or natural).Non-limiting examples of the non-proteinaceous moiety of the presentinvention include polyethylene glycol (PEG), Polyvinyl pyrrolidone(PVP), divinyl ether and maleic anhydride copolymer (DIVEMA; see forexample, Kaneda Y, et al., 1997, Biochem. Biophys. Res. Commun. 239:160-5) and poly(styrene comaleic anhydride) (SMA; see for example, Mu Y,et al., 1999, Biochem Biophys Res Commun. 255: 75-9).

Conjugation of such a non-proteinaceous moiety confers the polypeptideof this aspect of the present invention with stability (e.g., againstprotease activities) and/or solubility (e.g., within a biological fluidsuch as blood, digestive fluid) while preserving its biological activityand prolonging its half-life. Such a conjugation is advantageousparticularly in cases of therapeutic proteins which exhibit shorthalf-life and rapid clearance from the blood. The increased half-livesof conjugated proteins, in the plasma, results from increased size ofprotein conjugates (which limits their glomerular filtration) anddecreased proteolysis due to polymer steric hindrance. Generally, themore polymer chains attached per peptide, the greater the extension ofhalf-life. However, measures are taken not to reduce the specificactivity of the sFlt-14 amino acid sequence of the present invention(e.g., sFlt-14 binding to VEGF). Methods of conjugating non-proteinmoieties to amino acid sequences are well known in the art (as describedin, for example, Veronese F M, Biomaterials, Volume 22(5), 2001, pp.405-417(13), Elsevier Publishing; and Haruhiko Kamada, et al., 2000,Cancer Research 60: 6416-6420, which are fully incorporated herein byreference).

Thus, the present inventors have uncovered a novel soluble and secretedvariant of VEGFR. This variant is expressed in serum and placentae ofpreeclamptic subjects and as such detection of same may be clinicallyvaluable such as in the diagnosis of preeclampsia.

Thus, according to some embodiments of yet another aspect of the presentinvention there is provided a method of detecting sFlt-14 (e.g., SEQ IDNO: 2) in a biological sample (including in vivo detection).

Typically, the method is effected by determining sFlt-14 level, presenceor ratio (such as with respect to other Flt-1 isoforms, e.g., sFlt-1 asshown in FIG. 9B).

In accordance with some embodiments of this aspect of the presentinvention the methods comprising, contacting the biological sample withan antibody comprising an antigen recognition domain which specificallybinds the isolated polypeptide of sFlt-14 (e.g., SEQ ID NO: 2) and notto SEQ ID NO: 10 such that the sFlt-14 and the antibody form a complex;and measuring a presence or a level of the complex to thereby detectsFlt-14 in the biological sample.

As used herein a “biological sample” refers to a biological material,such as cells, tissues (e.g., placenta, chorionic villus sample, solidtumor) and fluids such as amniotic fluid, blood, serum, plasma, lymph,bile fluid, urine, saliva, sputum, synovial fluid, semen, tears,cerebrospinal fluid, bronchioalveolar large fluid, ascites fluid, pus,conditioned medium and the like in which sFlt-14 may be present. Thebiological sample is a maternal or fetal sample. The biological sample,may be ex vivo or in vitro analyzed, but can also be analyzed withoutretrieval from the subject's body.

As shown in Example 8 and in FIGS. 9A-B (in the Examples section whichfollows), sFlt-14 is exclusively expressed from week 13 of gestation.Thus, when analysis of sFlt-14 level or presence is effected prior tothis week, specific antibodies or oligonucleotides are preferablyemployed. From week 13 and on, the use of antibodies or oligonucleotidesdirected at common sequence regions of Flt-1 variants may also becontemplated.

Thus, antibodies of some embodiments of this aspect of the presentinvention may be directed to the amino acid sequence CELYTSTSPSSSSSS(SEQ. ID. NO: 5). This peptide comprises amino acids derived from theunique 28 amino acid sequence of sFlt-14 (SEQ. ID. NO: 4) which are notpresent in other Flt polypeptides (i.e. in the transmembrane and solublesFlt-1), as depicted in SEQ ID NO: 10. Alternatively, antibodies may bedirected to the amino acid sequence CHANGVPEPQITWFK (SEQ. ID. NO: 6).This peptide comprises amino acids derived from an amino acid sequenceshared by sFlt-14 and the transmembrane Flt-1, but not by the sFlt-1(SEQ ID NO: 10). Conversely, antibodies may be directed to the bridgingregion which comprises both the common amino acid sequence and theunique amino acid sequence. An exemplary bridging region whichantibodies can be directed to is HKIQQEPELYTSTS (SEQ. ID. NO: 15).Measures are taken to select antibodies which are specific to sFlt-14and not Flt-1 or its soluble form.

Specific peptides chosen for antibody generation are preferably selectedimmunogenic (i.e., capable of stimulating an antibody response).Parameters for testing peptide immunogenicity are well known in the artincluding, but not limited to, foreginess, molecular size, chemicalcomposition and heterogeneity and susceptibility to antigen processingand presentation. Various sequence analysis software applications areknown in the art, which provide an immunogenicity index according to,for example, the Jameson-Wolf algorithm. Examples include, but are notlimited to, Sciprot (available fromwwwdotasiaonlinedotnetdothk/˜twcbio/DOCS/1/scPrteindothtm) and Macvector(available from wwwdotaccelrysdotcom/products/macvector/) as well as thewidely utilized GCG package (Genetics Computer Group, Wisconsin).

The term “antibody” as used herein includes whole antibody molecules aswell as functional fragments thereof, such as Fab, F(ab′)₂, and Fv thatare capable of binding with antigenic portions of the targetpolypeptide. These functional antibody fragments constitute preferredembodiments of the present invention, and are defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule that can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(5) Single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain and the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule as described in, for example,U.S. Pat. No. 4,946,778.

Methods of generating such antibody fragments are well known in the art(See for example, Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York, 1988, incorporated herein byreference).

Purification of serum immunoglobulin antibodies (polyclonal antisera) orreactive portions thereof can be accomplished by a variety of methodsknown to those of skill in the art including, precipitation by ammoniumsulfate or sodium sulfate followed by dialysis against saline, ionexchange chromatography, affinity or immunoaffinity chromatography aswell as gel filtration, zone electrophoresis, etc. (see Goding in,Monoclonal Antibodies: Principles and Practice, 2nd ed., pp. 104-126,1986, Orlando, Fla., Academic Press). Under normal physiologicalconditions antibodies are found in plasma and other body fluids and inthe membrane of certain cells and are produced by lymphocytes of thetype denoted B cells or their functional equivalent. Antibodies of theIgG class are made up of four polypeptide chains linked together bydisulfide bonds. The four chains of intact IgG molecules are twoidentical heavy chains referred to as H-chains and two identical lightchains referred to as L-chains. Additional classes include IgD, IgE,IgA, IgM and related proteins.

Methods for the generation and selection of monoclonal antibodies arewell known in the art, as summarized for example in reviews such asTramontano and Schloeder, Methods in Enzymology 178, 551-568, 1989. AsFlt-14 polypeptide (or fragment thereof) of the present invention maybe used to generate antibodies in vitro. More preferably, the sFlt-14polypeptide of the present invention is used to elicit antibodies invivo. In general, a suitable host animal is immunized with the sFlt-14polypeptide of the present invention. Advantageously, the animal hostused is a mouse of an inbred strain. Animals are typically immunizedwith a mixture comprising a solution of the sFlt-14 polypeptide of thepresent invention in a physiologically acceptable vehicle, and anysuitable adjuvant, which achieves an enhanced immune response to theimmunogen. By way of example, the primary immunization conveniently maybe accomplished with a mixture of a solution of the sFlt-14 polypeptideof the present invention and Freund's complete adjuvant, the mixturebeing prepared in the form of a water in oil emulsion. Typically theimmunization will be administered to the animals intramuscularly,intradermally, subcutaneously, intraperitoneally, into the footpads, orby any appropriate route of administration. The immunization schedule ofthe immunogen may be adapted as required, but customarily involvesseveral subsequent or secondary immunizations using a milder adjuvantsuch as Freund's incomplete adjuvant. Antibody titers and specificity ofbinding to the sFlt-14 polypeptide can be determined during theimmunization schedule by any convenient method including by way ofexample radioimmunoassay, or enzyme linked immunosorbant assay, which isknown as the ELISA assay. When suitable antibody titers are achieved,antibody-producing lymphocytes from the immunized animals are obtained,and these are cultured, selected and cloned, as is known in the art.Typically, lymphocytes may be obtained in large numbers from the spleensof immunized animals, but they may also be retrieved from thecirculation, the lymph nodes or other lymphoid organs. Lymphocytes arethen fused with any suitable myeloma cell line, to yield hybridomas, asis well known in the art. Alternatively, lymphocytes may also bestimulated to grow in culture, and may be immortalized by methods knownin the art including the exposure of these lymphocytes to a virus, achemical or a nucleic acid such as an oncogene, according to establishedprotocols. After fusion, the hybridomas are cultured under suitableculture conditions, for example in multi-well plates, and the culturesupernatants are screened to identify cultures containing antibodiesthat recognize the hapten of choice. Hybridomas that secrete antibodiesthat recognize the sFlt-14 polypeptides of the present invention arecloned by limiting dilution and expanded, under appropriate cultureconditions. Monoclonal antibodies are purified and characterized interms of immunoglobulin type and binding affinity.

Antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment.

Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly. These methods are described, for example,by Goldenberg, in U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety (see also Porter, R. R., Biochem. J., 73: 119-126, 1959).Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Fv fragments comprise an association of V_(H) and V_(L) chains. Thisassociation may be noncovalent, as described in Inbar et al. (Proc.Nat'l Acad. Sci. USA 69:2659-62, 1972). Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise V_(H) and V_(L) chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the V_(H) and V_(L)domains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow andFilpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426,1988; Pack et al., Bio/Technology 11:1271-77, 1993; and Ladner et al.,U.S. Pat. No. 4,946,778, all of which are hereby incorporated, byreference, in entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick and FryMethods, 2: 106-10, 1991).

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include human immunoglobulins(recipient antibody) in which residues form a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies may also compriseresidues, which are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin [Jones et al., Nature, 321:522-525(1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr.Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source, which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human monoclonal antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

Thus the antibodies may be contacted with the biological sample so at toform an immunocomplex. Detection of the level and presence of thecomplex may be effected using methods which are well known in the art.Examples of such methods include, but are not limited to, Western blot,Radio-immunoassay (RIA), Fluorescence activated cell sorting (FACS), andImmunohistochemical analysis.

Alternatively, detection of sFlt-14 may be at the polynucleotide level.To this end, the sample is contacted with an isolated polynucleotide(e.g., oligonucleotide) which comprises a nucleic acid sequence whichspecifically binds to sFlt-14 (SEQ ID NO: 1 or 3 or to a bridgingsequence as, for example, as set forth in SEQ ID NO: 8) and not tosFlt-1 (SEQ ID NO: 9) so as to form a hybridization complex.

Following a sufficient time of incubation the presence or level of thecomplex is measured to thereby detect sFlt-14 in the biological sample.

For example, oligonucleotides can be used which are capable of bindingto sequences which are specific to sFlt-14 polynucleotides and not toother Flt-1 polynucleotides (e.g. membrane-anchored Flt-1 and solubleFlt-1) such as sFlt-1 (SEQ ID NO: 9). Such sequences may be present inthe untranslated region or the open reading frame of the isolatedpolynucleotides.

As used herein, the term “oligonucleotide” refers to a single-strandedor double-stranded oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally occurring bases, sugars, andcovalent internucleoside linkages (e.g., backbone), as well asoligonucleotides having non-naturally occurring portions, which functionsimilarly to respective naturally occurring portions.

As used herein, the phrase “capable of specifically hybridizing” refersto forming a double strand molecule such as RNA:RNA, RNA:DNA and/orDNA:DNA molecules.

Oligonucleotides designed according to the teachings of the presentinvention can be generated according to any oligonucleotide synthesismethod known in the art, such as enzymatic synthesis or solid-phasesynthesis. Equipment and reagents for executing solid-phase synthesisare commercially available from, for example, Applied Biosystems. Anyother means for such synthesis may also be employed; the actualsynthesis of the oligonucleotides is well within the capabilities of oneskilled in the art and can be accomplished via established methodologiesas detailed in, for example: Sambrook, J. and Russell, D. W. (2001),“Molecular Cloning: A Laboratory Manual”; Ausubel, R. M. et al., eds.(1994, 1989), “Current Protocols in Molecular Biology,” Volumes I-III,John Wiley & Sons, Baltimore, Md.; Perbal, B. (1988), “A Practical Guideto Molecular Cloning,” John Wiley & Sons, New York; and Gait, M. J., ed.(1984), “Oligonucleotide Synthesis”; utilizing solid-phase chemistry,e.g. cyanoethyl phosphoramidite followed by deprotection, desalting, andpurification by, for example, an automated trityl-on method or HPLC.

The oligonucleotide of the present invention is of at least 17, at least18, at least 19, at least 20, at least 22, at least 25, at least 30 orat least 40, bases specifically hybridizable with polynucleotidesequences of the present invention.

Hybridization based assays which allow the detection of a DNA or RNA ofinterest in a biological sample rely on the use of oligonucleotide whichcan be 10, 15, 20, or 30 to 100 nucleotides long preferably from 10 to50, more preferably from 40 to 50 nucleotides.

Hybridization of short nucleic acids (below 200 bp in length, e.g. 17-40bp in length) can be effected using the following exemplaryhybridization protocols which can be modified according to the desiredstringency; (i) hybridization solution of 6×SSC and 1% SDS or 3 M TMACI,0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100μg/ml denatured salmon sperm DNA and 0.1% nonfat dried milk,hybridization temperature of 1-1.5° C. below the T_(m), final washsolution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH7.6), 0.5% SDS at 1-1.5° C. below the T_(m); (ii) hybridization solutionof 6×SSC and 0.1% SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and0.1% nonfat dried milk, hybridization temperature of 2-2.5° C. below theT_(m), final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5° C. below the T_(m), finalwash solution of 6×SSC, and final wash at 22° C.; (iii) hybridizationsolution of 6×SSC and 1% SDS or 3 M TMACI, 0.01 M sodium phosphate (pH6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNAand 0.1% nonfat dried milk, hybridization temperature.

The detection of hybrid duplexes can be carried out by a number ofmethods. Typically, hybridization duplexes are separated fromunhybridized nucleic acids and the labels bound to the duplexes are thendetected. Such labels refer to radioactive, fluorescent, biological orenzymatic tags or labels of standard use in the art. A label can beconjugated to either the oligonucleotide probes or the nucleic acidsderived from the biological sample (target).

For example, oligonucleotides of the present invention can be labeledsubsequent to synthesis, by incorporating biotinylated dNTPs or rNTP, orsome similar means (e.g., photo-cross-linking a psoralen derivative ofbiotin to RNAs), followed by addition of labeled streptavidin (e.g.,phycoerythrin-conjugated streptavidin) or the equivalent. Alternatively,when fluorescently-labeled oligonucleotide probes are used, fluorescein,lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3,Cy3.5, Cy5, Cy5.5, Cy7, Fluor X (Amersham) and others [e.g., Kricka etal. (1992), Academic Press San Diego, Calif] can be attached to theoligonucleotides.

Traditional hybridization assays include PCR, RT-PCR, RNase protection,in-situ hybridization, primer extension, Northern Blot and dot blotanalysis (see Examples section hereinbelow).

It will be appreciated that detection of sFlt-14 may also be effected byother methods which do not include the use of antibodies oroligonucleotides. These methods, even if more laborious at time, includebut are not limited to molecular weight-based identification and massspectrometry.

It will be appreciated that the above described methods of detectingsFlt-14 is mostly desired for the diagnosis of a pregnancy associatedmedical condition associated with maternal or fetal stress.

As used herein the term “diagnosis” refers to classifying a disease or asymptom, determining a severity of such a disease, monitoring diseaseprogression, monitoring the effectiveness of a therapeutic regime,forecasting (prognosing) an outcome of a disease and/or prospects ofrecovery.

As used herein the phrase “a pregnancy associated medical conditionassociated with maternal or fetal stress” refers to a disease or asyndrome in which there are clinical symptoms in the mother of fetuswhich are associated with upregulation of sFlt-14. The pregnancy may beat any stage or phase. The medical condition may include anyhypertensive disorders: preeclampsia, eclampsia, mild preeclampsia,chronic hypertension, EPH gestosis, gestational hypertension,superimposed preeclampsia (including preeclampsia superimposed onchronic hypertension, chronic nephropathy or lupus), HELLP syndrome(hemolysis, elevated liver enzymes, low platelet count) or nephropathy.The medical condition may also include gestational diabetes, fetalgrowth restriction (FGR) and fetal alcohol syndrome (FAS).

As used herein, the phrase “maternal or fetal stress” refers to anycondition in which the mother or the fetus is at risk of developing apregnancy related complication. Fetal stress includes, without beinglimited to, inadequate nutrient supply and cessation of fetal growth.Maternal stress includes, without being limited to, hypertension anddiabetes. Fetal and maternal stress may affect fetal development andbrain functions and plays a significant role in pregnancy outcomesrelated to prematurity and urgent deliveries (e.g. c-section).

As used herein the phrase “subject in need thereof” refers to a mammalpreferably a human subject (e.g., pregnant female or a fetus).

Although this invention is described with respect to pregnant women,methods described herein may also be utilized to assess the risk tonon-pregnant women of developing hypertensive disorders duringpregnancy.

As mentioned herein above, the present inventors have shown that sFlt-14is significantly upregulated in preeclampsia (see Example 3 and FIGS.4A-B). Furthermore, the present inventors have shown that the novelVEGFR variant (sFlt-14) is expressed from early pregnancy (weeks 9) andis the dominant VEGFR starting from the second trimester (week 13, seeExample 8 and FIGS. 9A-B). Thus, the present inventors envision the useof agents capable of downregulating sFlt-14 for the treatment ofpregnancy associated medical conditions. In addition the presentinventors have successfully shown that the novel variant competes withVEGFR (see Example 9 and FIG. 10B) to binding of VEGF and as suchregulation of the novel variant is critical for the treatment ofVEGF-associated medical conditions.

As used herein “a VEGF associated medical condition” refers to adisease, disorder or condition which onset or progression of depend onreduced or excessive activity or expression of VEGFR ligands asdescribed above.

Thus, according to some embodiments of the present invention there isprovided a method of treating a VEGF-associated medical condition inwhich there is a reduced activity and/or expression of VEGF. The methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of an agent capable of downregulating sFlt-14 tothereby treat the VEGF-associated medical condition in the subject.

Examples of medical conditions associated with reduced activity and/orexpression of VEGF include, but are not limited to, preeclampsia,gestational diabetes, gestational hypertension, fetal growth restriction(FGR), fetal alcohol syndrome (FAS) and hypertension.

As used herein “treating” refers to preventing, curing, reversing,attenuating, alleviating, minimizing, suppressing or halting thedeleterious effects of a medical condition.

Examples of such agents include the above-described antibodies andpolynucleotides (e.g., capable of specifically binding and inhibitingsFlt-14 and not sFlt-1).

For example, the agent of this aspect of the present invention may becapable of reducing activity and/or expression (i.e. downregulating)sFlt-14 by affecting the cells which produce the sFlt-14 polypeptides(e.g. trophoblasts).

Thus, an agent capable of downregulating sFlt-14 of the presentinvention is an oligonucleotide capable of specifically hybridizing(e.g., in cells under physiological conditions) to a polynucleotidecomprising a nucleic acid sequence encoding a sFlt-14 polypeptide. Sucholigonucleotides have been described hereinabove.

Delivery strategies which can be used to efficiently deliveroligonucleotides into a wide variety of cell types are well known in theart [see, for example, Luft J Mol Med 76: 75-6 (1998); Kronenwett etal., Blood 91: 852-62 (1998); Rajur et al., Bioconjug Chem 8: 935-40(1997); Lavigne et al., Biochem Biophys Res Commun 237: 566-71 (1997)and Aoki et al., (1997) Biochem Biophys Res Commun 231: 540-5 (1997].

An example of an oligonucleotide agent capable of downregulating theexpression of sFlt-14 polypeptides is a small interfering RNA (siRNA)molecule. RNA interference is a two-step process. During the first step,which is termed the initiation step, input dsRNA is digested into 21-23nucleotide (nt) small interfering RNAs (siRNA), probably by the actionof Dicer, a member of the RNase III family of dsRNA-specificribonucleases, which cleaves dsRNA (introduced directly or via anexpressing vector, cassette or virus) in an ATP-dependent manner.Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNA),each strand with 2-nucleotide 3′ overhangs [Hutvagner and Zamore Curr.Opin. Genetics and Development 12:225-232 (2002); and Bernstein Nature409:363-366 (2001)].

In the effector step, the siRNA duplexes bind to a nuclease complex toform the RNA-induced silencing complex (RISC). An ATP-dependentunwinding of the siRNA duplex is required for activation of the RISC.The active RISC then targets the homologous transcript by base pairinginteractions and cleaves the mRNA into 12 nucleotide fragments from the3′ terminus of the siRNA [Hutvagner and Zamore Curr. Opin. Genetics andDevelopment 12:225-232 (2002); Hammond et al., (2001) Nat. Rev. Gen.2:110-119 (2001); and Sharp Genes. Dev. 15:485-90 (2001)]. Although themechanism of cleavage is still to be elucidated, research indicates thateach RISC contains a single siRNA and an RNase [Hutvagner and ZamoreCurr. Opin. Genetics and Development 12:225-232 (2002)].

Because of the remarkable potency of RNAi, an amplification step withinthe RNAi pathway has been suggested. Amplification could occur bycopying of the input dsRNAs, which would generate more siRNAs, or byreplication of the siRNAs formed. Alternatively or additionally,amplification could be effected by multiple turnover events of the RISC[Hammond et al., Nat. Rev. Gen. 2:110-119 (2001), Sharp Genes. Dev.15:485-90 (2001); Hutvagner and Zamore Curr. Opin. Genetics andDevelopment 12:225-232 (2002)]. For more information on RNAi see thefollowing reviews Tuschl ChemBiochem. 2:239-245 (2001); Cullen Nat.Immunol. 3:597-599 (2002); and Brantl Biochem. Biophys. Act. 1575:15-25(2002).

Synthesis of RNAi molecules suitable for use with the present inventioncan be effected as follows. First, the sFlt-14 polynucleotide sequencetarget is scanned downstream for AA dinucleotide sequences. Occurrenceof each AA and the 3′ adjacent 19 nucleotides is recorded as potentialsiRNA target sites.

Second, potential target sites are compared to an appropriate genomicdatabase (e.g., human, mouse, rat etc.) using any sequence alignmentsoftware, such as the BLAST software available from the NCBI server(wwwdotncbidotnlmdotnihdotgov/BLAST/). Putative target sites thatexhibit significant homology to other coding sequences are filtered out.

Qualifying target sequences are selected as template for siRNAsynthesis. Preferred sequences are those including low G/C content asthese have proven to be more effective in mediating gene silencing ascompared to those with G/C content higher than 55%. Several target sitesare preferably selected along the length of the target gene forevaluation. For better evaluation of the selected siRNAs, a negativecontrol is preferably used in conjunction. Negative control siRNApreferably include the same nucleotide composition as the siRNAs butlack significant homology to the genome. Thus, a scrambled nucleotidesequence of the siRNA is preferably used, provided it does not displayany significant homology to any other gene.

Other nucleic acid agents which can be used to downregulate expressionof sFlt-14 include but are not limited to a DNAzyme molecule capable ofspecifically cleaving its encoding polynucleotide. DNAzymes aresingle-stranded polynucleotides which are capable of cleaving bothsingle and double stranded target sequences (Breaker, R. R. and Joyce,G. Chemistry and Biology 1995; 2: 655; Santoro, S. W. & Joyce, G. F.Proc. Natl, Acad. Sci. USA 1997; 94:4262); a ribozyme molecule capableof specifically cleaving its encoding polynucleotide. Ribozymes arebeing increasingly used for the sequence-specific inhibition of geneexpression by the cleavage of mRNAs encoding proteins of interest [Welchet al., Curr Opin Biotechnol. 9:486-96 (1998)]. The possibility ofdesigning ribozymes to cleave any specific target RNA has rendered themvaluable tools in both basic research and therapeutic applications; atriplex forming oligonucleotides (TFOs). In the last decade, studieshave shown that TFOs can be designed which can recognize and bind topolypurine/polypirimidine regions in double-stranded helical DNA in asequence-specific manner. Thus the DNA sequence encoding the polypeptideof the present invention can be targeted thereby down-regulating thepolypeptide.

Downregulating sFlt-14 can also be effected at the protein level.

Thus, another example of an agent capable of downregulating apolypeptide of the present invention is an antibody or antibody fragmentcapable of specifically binding sFlt-14 or a homologue thereof,preferably to its active site, thereby preventing its function. Methodsof producing such antibodies are described hereinabove.

Regardless of the agents employed, the effect of same on sFlt-14activity and/or expression (and indirectly VEGF activity or expression)may be determined using well known molecular biology, biochemical orcell biology techniques. The specific assay will be selected accordingto the particular researcher's needs and expertise.

As mentioned hereinabove, soluble VEGF receptors bind and antagonizeVEGF activity. Indeed, the present inventors have further shown thatsFlt-14 antagonizes VEGF (Example 9 and FIG. 10B).

Thus, according to some embodiments of the present invention there isprovided a method of treating a VEGF-associated medical condition inwhich there is an excessive activity and/or expression of VEGF (such anexcessive activity or expression often results in hyperangiogenesis).The method comprising administering to a subject in need thereof atherapeutically effective amount of an agent capable of upregulatingsFlt-14 to thereby treat the VEGF-associated medical condition in thesubject.

Examples of medical conditions associated with excessive activity and/orexpression of VEGF include, but are not limited to, cancer, eyedisorders such as neovascularization of the cornea, polycystic ovarydisease and endometriosis.

As such, the present invention envisions use of the novel sFlt-14 forantagonizing VEGF activity and thereby reducing angiogenesis which maybe harnessed for the treatment of VEGF associated conditions (e.g.,cancer).

As used herein the term “angiogenesis” refers to the production ordevelopment of blood vessels.

As used herein the term “cancer” refers to any tumoral disease includingmetastasis. Examples of cancer include but are not limited to carcinoma,lymphoma, blastoma, sarcoma, and leukemia. Particular examples ofcancerous diseases but are not limited to: Myeloid leukemia such asChronic myelogenous leukemia. Acute myelogenous leukemia withmaturation. Acute promyelocytic leukemia, Acute nonlymphocytic leukemiawith increased basophils, Acute monocytic leukemia. Acute myelomonocyticleukemia with eosinophilia; Malignant lymphoma, such as Birkitt'sNon-Hodgkin's; Lymphoctyic leukemia, such as Acute lymphoblasticleukemia. Chronic lymphocytic leukemia; Myeloproliferative diseases,such as Solid tumors Benign Meningioma, Mixed tumors of salivary gland,Colonic adenomas; Adenocarcinomas, such as Small cell lung cancer,Kidney, Uterus, Prostate, Bladder, Ovary, Colon, Sarcomas, Liposarcoma,myxoid, Synovial sarcoma, Rhabdomyosarcoma (alveolar), Extraskeletelmyxoid chonodrosarcoma, Ewing's tumor; other include Testicular andovarian dysgerminoma, Retinoblastoma, Wilms' tumor, Neuroblastoma,Endometrial cancer, Malignant melanoma, Mesothelioma, breast, skin,prostate, and ovarian.

As used herein the phrase “neovascularized cornea” refers to theabnormal, pathological condition in which the cornea becomes vascular.

Other medical conditions, diseases and disease processes in whichangiogenesis plays a role can be treated according to the teachings ofthe present invention. These include, but are not limited to, diabeticretinopathy, neovascular glaucoma, rheumatoid arthritis and hemangiomas.

Agents capable of upregulating the polypeptides of the presentinvention, which may be used for the treatment of VEGF associatedconditions (e.g., cancer or corneal neovascularization), comprise theisolated polypeptides per se or polynucleotides of the presentinvention.

Thus, polynucleotides of the present invention can be administered tothe subject employing any suitable mode of administration, describedhereinbelow (i.e., in vivo gene therapy). Alternatively, the nucleicacid construct is introduced into a suitable cell via an appropriategene delivery vehicle/method (transfection, transduction, homologousrecombination, etc.) and an expression system as needed and then themodified cells are expanded in culture and returned to the individual(i.e., ex vivo gene therapy).

Such polynucleotide sequences are typically inserted into expressionvectors to enable expression of the recombinant polypeptide. Theexpression vector of the present invention includes additional sequenceswhich render this vector suitable for replication and integration inprokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).Typical cloning vectors contain transcription and translation initiationsequences (e.g., promoters, enhances) and transcription and translationterminators (e.g., polyadenylation signals).

To enable cellular expression of the polynucleotides of the presentinvention, the nucleic acid construct of the present invention furtherincludes at least one cis acting regulatory element. As used herein, thephrase “cis acting regulatory element” refers to a polynucleotidesequence, preferably a promoter, which binds a trans acting regulatorand regulates the transcription of a coding sequence located downstreamthereto.

Any suitable promoter sequence can be used by the nucleic acid constructof the present invention.

Preferably, the promoter utilized by the nucleic acid construct of thepresent invention is active in the specific cell population transformed.Examples of cell type-specific and/or tissue-specific promoters includepromoters such as albumin that is liver specific [Pinkert et al., (1987)Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al.,(1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cellreceptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins;[Banerji et al. (1983) Cell 33729-740], neuron-specific promoters suchas the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad.Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al.(1985) Science 230:912-916] or mammary gland-specific promoters such asthe milk whey promoter (U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). The nucleic acid construct of the presentinvention can further include an enhancer, which can be adjacent ordistant to the promoter sequence and can function in up regulating thetranscription therefrom.

The nucleic acid construct of the present invention may further includean appropriate selectable marker and/or an origin of replication. Forexample, the nucleic acid construct utilized may be a shuttle vector,which can propagate both in E. coli (wherein the construct comprises anappropriate selectable marker and origin of replication) and becompatible for propagation in cells, or integration in a gene and atissue of choice. The construct according to the present invention canbe, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, avirus or an artificial chromosome.

In addition to the elements already described, the expression vector ofthe present invention may typically contain other specialized elementsintended to increase the level of expression of cloned nucleic acids orto facilitate the identification of cells that carry the recombinantDNA. For example, a number of animal viruses contain DNA sequences thatpromote the extra chromosomal replication of the viral genome inpermissive cell types. Plasmids bearing these viral replicons arereplicated episomally as long as the appropriate factors are provided bygenes either carried on the plasmid or with the genome of the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryoticreplicon is present, then the vector is amplifiable in eukaryotic cellsusing the appropriate selectable marker. If the vector does not comprisea eukaryotic replicon, no episomal amplification is possible. Instead,the recombinant DNA integrates into the genome of the engineered cell,where the promoter directs expression of the desired nucleic acid.

Examples of mammalian expression vectors include, but are not limitedto, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay,pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1,pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Strategene, pTRES which is available from Clontech, andtheir derivatives.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used. SV40 vectors includepSVT7 and pMT2. Vectors derived from bovine papilloma virus includepBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, andp2O5. Other exemplary vectors include pMSG, pAV009/A+, pMTO10/A+,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the SV-40 early promoter, SV-40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or other promotersshown effective for expression in eukaryotic cells.

Recombinant viral vectors may also be used to synthesize thepolynucleotides of the present invention. Viruses are very specializedinfectious agents that have evolved, in many cases, to elude hostdefense mechanisms. Typically, viruses infect and propagate in specificcell types. The targeting specificity of viral vectors utilizes itsnatural specificity to specifically target predetermined cell types andthereby introduce a recombinant gene into the infected cell. Bone marrowcells can be targeted using the human T cell leukemia virus type I(HTLV-I).

Currently preferred in vivo nucleic acid transfer techniques includetransfection with viral or non-viral constructs, such as adenovirus,lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) andlipid-based systems. Useful lipids for lipid-mediated transfer of thegene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al.,Cancer Investigation, 14(1): 54-65 (1996)]. The most preferredconstructs for use in gene therapy are viruses, most preferablyadenoviruses, AAV, lentiviruses, or retroviruses. A viral construct suchas a retroviral construct includes at least one transcriptionalpromoter/enhancer or locus-defining element(s), or other elements thatcontrol gene expression by other means such as alternate splicing,nuclear RNA export, or post-translational modification of messenger.Such vector constructs also include a packaging signal, long terminalrepeats (LTRs) or portions thereof, and positive and negative strandprimer binding sites appropriate to the virus used, unless it is alreadypresent in the viral construct. In addition, such a construct typicallyincludes a signal sequence for secretion of the peptide from a host cellin which it is placed. Preferably the signal sequence for this purposeis a mammalian signal sequence or the signal sequence of the polypeptidevariants of the present invention. Optionally, the construct may alsoinclude a signal that directs polyadenylation, as well as one or morerestriction sites and a translation termination sequence. By way ofexample, such constructs will typically include a 5′ LTR, a tRNA bindingsite, a packaging signal, an origin of second-strand DNA synthesis, anda 3′ LTR or a portion thereof. Other vectors can be used that arenon-viral, such as cationic lipids, polylysine, and dendrimers.

Various methods can be used to introduce the expression vector of thepresent invention into cells. Such methods are generally described inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringsHarbor Laboratory, New York (1989, 1992), in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich.(1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995),Vectors: A Survey of Molecular Cloning Vectors and Their Uses,Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4(6): 504-512, 1986] and include, for example, stable or transienttransfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

Introduction of nucleic acids by viral infection offers severaladvantages over other methods such as lipofection and electroporation,since higher transfection efficiency can be obtained due to theinfectious nature of viruses.

It will be appreciated that up-regulating the expression and/or functionof a sFlt-14 polypeptide will typically result in a reduced VEGFactivity.

The above described agents of the present invention can be provided tothe individual per se, or as part of a pharmaceutical composition whereit is mixed with a pharmaceutically acceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the isolated polypeptides,the isolated polynucleotides, or the antibody preparations, which areaccountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases. One of the ingredients included in thepharmaceutically acceptable carrier can be for example polyethyleneglycol (PEG), a biocompatible polymer with a wide range of solubility inboth organic and aqueous media (Mutter et al. (1979).

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, transnasal, intestinal or parenteral delivery,including intramuscular, subcutaneous and intramedullary injections aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the preparation in a local rather thansystemic manner, for example, via injection of the preparation directlyinto a specific region of a patient's body.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological saltbuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, for oralingestion by a patient. Pharmacological preparations for oral use can bemade using a solid excipient, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The preparations described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The preparation of the present invention may also be formulated inrectal compositions such as suppositories or retention enemas, using,e.g., conventional suppository bases such as cocoa butter or otherglycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients effective to prevent, alleviate or amelioratesymptoms of disease or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro assays. For example, a dose can be formulated in animal modelsand such information can be used to more accurately determine usefuldoses in humans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. [See e.g., Fingl, et al., (1975) “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1].

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions including the preparation of the present inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert.

It is expected that during the life of a patent maturing from thisapplication many relevant polypeptides and polynucleotides encodingsFlt-14 will be developed and the scope of the term polypeptides andpolynucleotides encoding sFlt-14 is intended to include all such newtechnologies a priori.

As used herein the term “about” refers to ±10%

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”. This termencompasses the terms “consisting of” and “consisting essentially of”.

The phrase “consisting essentially of” means that the composition ormethod may include additional ingredients and/or steps, but only if theadditional ingredients and/or steps do not materially alter the basicand novel characteristics of the claimed composition or method.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate some embodiments of the invention in anon limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Sequence of the Novel sFlt-14

Materials and Experimental Procedures

RNA

Placental tissue was homogenized with a Polytron homogenizer andpreceded the total RNA extraction in TRI Reagent (Sigma) according tothe manufacturer's protocol. Cells were harvested and RNA was extractedin TRI Reagent.

RACE

Rapid Amplification of cDNA Ends was preformed using BD SMART™ RACE cDNAamplification kit. 3′ RACE based on preeclamptic placental RNA was usedwith a primer taken from the beginning of FLT1's exon 14 used as the 5′primer CCTCCTGCGAAACCTCAGTG (SEQ ID NO: 12) and a 3′ primer that wassupplied with the RACE kit: AAGCAGTGGTATCAACGCAGAGTAC(T)₃₀ VN (SEQ IDNO: 11).

Results

As is illustrated in FIGS. 1-3, the novel sFlt1 of the present inventiondiffers from the full transmembrane receptor Flt1 and from the knownsFlt1 in the following three aspects:

1) sFlt-14 does not comprise the 31 amino acids unique to sFlt-1(derived from intron 13) as clear from FIG. 1 (marked by the lightcoloring).2) sFlt-14 contains RNA sequences, as well as an amino acid stretch, notpresent in sFlt-1. The amino acid sequence includes amino acids derivedfrom exon 14 (which are also present in the full transmembrane receptor,see FIG. 1) as well as a unique amino acid sequence derived from intron14 (marked by the light coloring, see FIG. 1). As shown in FIG. 3A, thisunique sequence comprises a stretch of 28 amino acids derive from intron14.3) As shown in FIG. 2A, sFlt-14 also contains a unique regulatorysequence, namely a 3′-UTR fully derived from intron 14, including an Aluelement contained within its 3′-UTR.

Furthermore, as is illustrated in FIG. 2A, the cDNA sequence that isunique to the novel sFlt-14 comprises sequences not present in cDNAsfrom either the full transmembrane Flt1 or from the soluble sFlt-1. Asearch throw database revealed several EST sequences deposited in databases where only fragments of this unique transcript were identified(FIG. 2B). However, this alternatively-spliced sequence variance of Flt(namely sFlt-14 of the present invention) was not mentioned previouslyin the published literature, let alone association with any tissue orpathology.

Example 2 Generation of sFlt-14 Specific Antibodies

Materials and Experimental Procedures

Generation of sFlt-14 Specific Polyclonal Antibodies

Polyclonal antibodies were generated as described in the Sigma-Aldrich'sprotocol. In short, two peptides derived from sFlt-14 were synthesized(CHFK, SEQ ID NO: 6 and CESS, SEQ ID NO: 5) and injected into rabbits inorder to produce anti sFlt-14 sera. Three injections for each peptidewere performed, with a month period kept between injections. At the endof this procedure rabbit serums were evaluated for sFlt-14 reactivity.

Results

Two short peptides were generated from the amino acid sequence whichdistinguishes the novel sFlt-14 of the present invention from thepreviously described sFlt-1. As illustrated in FIG. 3B, the firstpeptide, termed CHFK (SEQ ID NO: 6), which was derived from exon 14, isnot comprised in sFlt-1 but is present in the full transmembranereceptor. The second peptide, termed CESS (SEQ ID NO: 5), was derivedfrom a sequence unique only to the novel sFlt-14 (and is not comprisedin either the transmembrane Flt-1 or sFlt-1). These two peptides wereused to elicit polyclonal antibodies as described in the experimentaldescription hereinabove.

The polyclonal antibodies generated can distinguish between the novelsFlt-14 and the previously described sFlt-1.

Example 3 Relative Abundance of Transmembrane Flt-1, sFlt-1 and sFlt-14in Different Cells

Materials and Experimental Procedures

Cells

Cells were obtained and cultured as previously described in Gluzman etal. [Gluzman et al., Biochem Biophys Res Commun. (2007) 359:263-8].

RNA

Normal placenta tissue and preeclampsia placenta tissue were homogenizedwith a Polytron homogenizer and preceded the total RNA extraction in TRIReagent (Sigma) according to the manufacturer's protocol. Cells wereharvested and RNA was extracted in TRI Reagent.

Northern Blotting

Total RNA (5-20 μg) was resolved by formaldehyde—agarose (1%) denaturinggels and blotted to positively charged nylon membrane by capillaryelution. The RNA was UV crosslinked (1200 j/m2) and the membrane wasstained with 0.1% methylene blue to ensure equal loading and transfer.Blots were hybridized overnight with a ³²P-labeled probe by a rediprimekit (Amersham). The blots were subjected to two washes (with 2×SSC, 1%SDS) for 30 minute at 60° C., after which they were exposed to MSsensitive film (Kodak). Three different probes from different regions ofthe FLT1 transcripts were used in order to allow detection of thevarious FLT1 isoforms: 1) an extra-cellular coding region for thedetection of all the isoforms; 2) an intron 14 region for the detectionof sFLT-14; and 3) an intron 13 region for the detection of sFLT1.

Results

As illustrated in FIG. 4A, RNA obtained from HUVEC, normal placenta andpreeclampsia placenta were separated on an RNA blot and hybridized witha probe common to Flt-1, sFlt-1, and sFlt-14 (which were distinguishedby the band position).

As evident from the results, in endothelial cells, known as the‘traditional’ cells expressing VEGF receptors, the predominant solubleFlt is sFlt-1 (also termed herein sFlt-13). In sharp contrast, inplacenta the predominant soluble receptor is the novel sFlt-14 of thepresent invention. The identity of the respective splice variant wasfurther validated through re-probing with probes specific for sFlt1 andfor sFlt-14 (data not shown).

Likewise, a comparison between endothelial cells and vascular smoothmuscle cells shows that whereas endothelial cells predominantly expresssFlt-1, vascular smooth muscle cells predominantly express the novelsFlt-14 (FIG. 4B). sFlt-14 has also been demonstrated as thepredominant, if not the exclusive, Flt isoform present in trophoblastsand dendritic cells (data not shown). Taken together these resultsindicate that sFlt-14 may be viewed as non-endothelial cell-specific,whereas, sFlt1 as the variant present in endothelial cells.

It is important to note that since alternative splicing which generatesthe full receptor or, alternatively, the soluble receptor (eithervariant) are mutually exclusive, the following occurs: In case sFlt1 isgenerated (e.g. by endothelial cells), the ratio of membrane-spanningFlt1 receptor to soluble Flt1 receptor is high. In contrast, thealternative splicing mode which generates sFlt-14, allows the dominancyof sFlt14 over the membrane spanning Flt1 receptor. This results in anet transition from pro- to anti-VEGF signaling and renders theexpressing cells irresponsive to VEGF.

Example 4 In Situ Protein and mRNA Detection of sFlt-14 in PreeclamticPlacenta

Materials and Experimental Procedures

In Situ Hybridization

Placental paraffin embedded sections were hybridized with a S35riboprobe taken from the intron 14 region of sFlt-14 (SEQ ID NO: 13) aswas previously described by Motro et al. [Motro et al., PNAS, 1990,87(8), 3092-6].

Immunohistochemistry

A sFlt-14 specific rabbit polyclonal antibody at a 1:100 dilution wasused on paraffin embedded placental sections. Antigen retrieval wascarried out using 25 mM citrate buffer pH=6.0. The antibody was directedagainst a peptide derived from the C-terminus of the sFLT-14protein—CELYTSTSPSSSSSS (CESS antibody, SEQ ID NO: 5).

Results

FIGS. 5A-B demonstrate the major value of the CESS antibody directedspecifically against the unique section of sFlt-14 (the amino acids ofintron 14) as illustrated in immunohistological detection of sFlt-14proteins in placental sections. Likewise, FIGS. 5C-D demonstrate theimportance of the unique mRNA probe (complementary to the unique intron14 sequence) for specific detection of sFlt-14 mRNA as illustrated by insitu hybridization of placental sections with the specific probe. Itshould be emphasized that there is no cross-reaction of the CESSantibody or of the probe with the full receptor Flt and the solublesFlt-1 so that the reagents are truly exclusive for the novel sFlt-14.

FIGS. 5A-D also provide some mechanistic insights to the pathogenicprocess as these results identify, for the first time, which cells inthe diseased placenta produce the soluble receptor (cells that were notidentified using the known sFlt1). These findings illustrate thattrophoblastic cells within the syncytial knots produce the solublesFlt-14. These results are consistent with the fact that syncytial knotsare much more abundant in pre-eclampsia compared to normal pregnancy,and are a hallmark of a degenerative placenta.

Example 5 Expression of sFlt-14 in Normal Term Placentae

Materials and Experimental Procedures

Western Blotting

Three normal term placentae were homogenized and separated into twogroups: group 1) subjected to a pre immune cleaning treatment; and group2) not subjected to cleaning treatment. The cleaning treatment (used toclean the sample from proteins that might interact with the irrelevantantibodies of the CESS serum in the detection step) included 3 hourincubation with 20 μl rabbit pre-immune serum followed by an addition ofProtein A beads (P3391, Sigma), overnight incubation and precipitation.The non-cleaned treatment was the same, without the addition of rabbitpre-immune serum.

Each sample, cleaned or non-cleaned, was separated into two differentimmunoprecipitations, one with the CESS antibody and the other with theFLT11 antibody (V4262, Sigma). The precipitants were loaded on a 6%acrylamide gel, run electrophoretically, transferred to a membrane anddetected with the CESS antibody.

Results

As clearly illustrated in FIG. 6, the size of the sFLT14 protein isapproximately 110 Kd. It is precipitated by the FLT11 antibody, whichtargets the extracellular domain of Flt-1, and visualized by the CESSantibody, which specifically targets the C′ terminus of the novelsFlt-14, validating the existence of a novel variant of the solubleFlt1. These results also proved that the unique CESS epitope is anintegral part of a splice variant that includes the extracellularbinding domain of Flt1.

Example 6 Mass-Spectrometry Identification of sFLT14

Materials and Experimental Procedures

Mass Spectrometry

A preeclamptic placenta was homogenized in a protein lysis buffer, andincubated for 3 hours with 20 μl rabbit pre-immune serum (in order toclean the sample from proteins that might interact with the irrelevantantibodies of the CESS serum). Protein A beads (P3391, Sigma) were addedfor overnight incubation and precipitated. 15 μl CESS antibody was addedto the cleaned homogenate, incubated for 3 hours, followed by anaddition of protein A beads and another overnight incubation. The beadswere precipitated, washed, and boiled with sample buffer. The sampleswere loaded on a 6% acrylamide gel and run electrophoretically. The gelwas stained with coomassie blue and destained till bands appeared. A 110Kd band was cut for mass spectrometry analysis. The band was digested bytrypsin, analyzed by LC-MS/MS on DECA/LCQ and identified by Pep-Minerand Sequest software against nr database of human, mouse, rat, bovineand rabbit.

Results

As depicted in FIG. 7, a positive identification of Flt1 was achieved bythree peptides taken from the extracellular domain of Flt 1. Theseresults proved that the extracellular domain of Flt1 and the C′ terminusof the novel sFlt-14 are on the same continuity. Eight amino-acids ofthe DQEAPYLLR peptide (QEAPYLLR, SEQ ID NO. 19) are encoded by exon 14,further strengthening the inclusion of exon 14 in the novel C′ terminus.

Example 7 Presence of sFlt-14 Isoforms in Serum of Preeclamptic Subjects

Materials and Experimental Procedures

Analysis of Serum Proteins

20 ml serum samples from PE subjects were concentrated via capture onFLT11-coated beads and elution. Affinity-purified proteins were analyzedby Western-blotting. Accordingly, proteins were separated on 6%acrylamide gel, electrophoretically transferred to a membrane, andimmunoblotted with the sFlt-14 specific CESS antibody. Protein detectionwas carried out using CESS antibody or the ab9540 antibody.

Results

Following the novel results illustrating that PE placentae expressupregulated levels of sFlt-14 (Examples 3 and 4 hereinabove), inventorsof the present invention investigated which sFlt isoform accumulates inthe serum of PE subjects. To identify the characteristics of circulatingsoluble receptors, inventors of the present invention analyzedpreeclamptic serum specimens. This was done by affinity purifying sFltisoforms from serum of PE patients with the FLT11 extracellularantibody. The purified isoforms were western blotted next to a CESSimmunoprecipitate of the placenta using the specific sFlt-14 antibody(CESS antibody). As illustrated in FIG. 8, the same two protein bandswere detected in the placenta and serum of PE subjects. These twoproteins were previously identified as sFlt-14 proteins (Example 3,hereinabove). Furthermore, the sFlt-14 protein detected in the serum ofPE subjects was visualized as two bands identical in size to thoseproduced by cells transfected with sFlt-14 expression plasmid (seeExample 10 hereinbelow) and detected with the sFlt-14-specific antibody.A second immunoblotting using the ab9540 extracellular targetingantibody failed to give different bands than the two mentioned above(data not shown), thus eliminating sFlt-1 existence in the PE serumsthat were tested, indicating that sFlt-14 is the major VEGF receptor inthe circulation of PE subjects.

Example 8 sFLT-14 is the Exclusive sFLT1 Isoform from the SecondTrimester of Pregnancy

Materials and Experimental Procedures

Northern Blotting

Total RNA was generated from human placental biopsies at different timepoints of gestation (weeks 9-11, 13 and 39 of gestation). 10-15 μg RNAof each sample was resolved by formaldehyde—agarose (1%) denaturing gelsand blotted to positively charged nylon membrane by capillary elution.The RNA was UV crosslinked (1200 j/m2) and the membrane was stained with0.1% methylene blue to ensure equal loading and transfer. Blots werehybridized overnight with a ³²P-labeled probe by a rediprime kit(Amersham). The probe used (SEQ ID NO: 14) targeted the shared sequencesof both isoforms, thus able to show their relative abundance. The blotswere subjected to two washes (with 2×SSC, 1% SDS) for 30 minute at 60°C., after which they were exposed to MS sensitive film (Kodak).

Results

In order to characterize the relative contribution of each of the sFLT1isoforms during the normal course of gestation, RNA was generated fromhuman placental biopsies at different time points during the course ofnormal gestation and an extracellular probe targeting the sharedsequences of both isoforms was used for hybridization (see materials andexperimental section above). As illustrated in FIGS. 9A-B, weeks 9-11 ofgestation (first trimester) are characterized by a 1:1 ratio of thesFlt-1 and sFlt-14 isoforms. However, at week 13 of gestation (beginningof the second trimester) sFlt-14 becomes the dominant, if not theexclusive isoform expressed in placentae. Furthermore, at week 39 (thirdtrimester) sFlt-14 remains the exclusive isoform. The exclusiveexpression of sFLT-14 from the second trimester of pregnancy and onwardcorresponds to the fact that sFLT-14 is significantly upregulated inpreeclampsia (a condition that usually occurs during the third trimesterof pregnancy).

Example 9 sFLT-14 is a Potent VEGF Inhibitor

Materials and Experimental Procedures

Expression of Recombinant sFlt1 and sflt-14 Proteins in Hela Cells

cDNAs encompassing the entire coding region of both soluble receptorisoforms sFlt-14 and sFlt-1 (SEQ ID NOs: 1 and 9, respectively) weresub-cloned into Bluescript expression vectors and transfected into T7polymerase-expressing human Hela cells. 20-24 hours later, growth mediawere collected and cells were harvested. Secreted proteins and cellassociated proteins were immunoprecipitated with the FLT11 antibody(V4262, Sigma) and analyzed by immunoblotting with antibodies (Ab9540,Abcam) directed against the extracellular domain of both sFlt-1 andsFlt-14.

ELISA

Analysis of sFlt-1 and sFlt-14 secretion of into the growth medium wascarried out using ELISA directed against a shared extracellular epitopeusing DVR100 (R&D systems).

Immunoprecipitation and Western Blotting

sFlt-1 and sFlt-14 secretion into the growth medium was analyzed byimmunoprecipitation with the FLT11 antibody and western immunoblottingwas carried out with the ab9540 antibody. Western blotting was furthercarried out as described in Example 5.

VEGF Inhibition Assay

Porcine Aortic Endothelial (PAE) cells engineered to express high levelsof human VEGF-R2 were acquired from Prof. Gera Neufeld (Technion, Haifa,Israel). Cells were grown in 10% FCS DMEM growth medium.

Increasing amounts of sFlt-14 or sFlt-1 (20, 40, 80 ng/ml) werepre-incubated with a constant amount of VEGF (20 ng/ml) prior to addingthe growth medium of PAE cells. VEGF-R2 phosphorylation levels weremeasured as a function of added sFlt-14/VEGF ratio or sFlt-1/VEGF ratio.A reduction in VEGF-R2 phosphorylation was determined using antibodiesdetecting phospho-VEGF-R2 (Cell-signaling, Cat. #2478) and standardizedto total VEGF-R2 protein visualized by immunoblotting with anti-VEGF-R2antibody (Santa Cruz Cat. SC-504).

Results

Since sFlt1 and sFlt-14 are qualitatively different proteins (sFlt-14contains 75 amino acids not present in sFlt1 and sFlt1 contains 31highly-conserved amino acids not present in sFlt-14), inventors of thepresent invention wished to demonstrate that sFlt-14 is in fact a VEGFreceptor capable of specifically binding and antagonizing VEGF.

To this end, inventors generated sFlt-14 expressing human Hela cellsand, for comparison, generated sFlt1-expressing Hela cells. ELISAanalysis (directed against a shared extracellular epitope) has indicatedthat the secretion of sFlt-1 and sFlt-14 into the respective growthmedium was comparable (concentrations of 100-200 ng/ml were detected forboth, data not presented). Furthermore, inventors confirmed the mutuallyexclusive presence of either sFlt-14 or sFlt-1 in the respective growthmedia by immunoprecipitation and western blots, as evident by theapparent molecular size of the immunoreactive protein (130 Kd and 120Kd, respectively, FIG. 10A).

To determine whether sFlt-14 inhibits VEGF signaling, increasing amountsof sFlt-14 were pre-incubated with a constant amount of VEGF (20 ng/ml)prior to adding the growth medium of Porcine Aortic Endothelial (PAE)cells engineered to express high levels of human VEGF-R2. VEGF-R2phosphorylation levels were measured as a function of added sFlt-14/VEGFratio. As shown in FIG. 10B, nearly complete inhibition of VEGF-R2phosphorylation was evident already at a 1:1 sFlt-14/VEGF ratio.Conversely, at this ratio, sFlt1 did not significantly inhibit VEGF-R2phosphorylation and, in fact, inhibited VEGF-R2 phosphorylation only athigher sFlt-1/VEGF ratios. Taken together, these results conclusivelyshowed that sFlt-14 is a potent inhibitor of VEGF signaling and notablymore potent than sFlt-1.

Example 10 sFlt-14 is Expressed in Human Cornea

Materials and Experimental Procedures

Immunohistochemistry

Corneal sections were isolated from human corneas that were removed dueto a diseased state. A sFlt-14 specific rabbit polyclonal antibody at a1:100 dilution was used on paraffin embedded corneal sections. Antigenretrieval was carried out using 25 mM citrate buffer pH=6.0. Theantibody was directed against a peptide derived from the C-terminus ofthe sFlt-14 protein—CELYTSTSPSSSSSS (CESS antibody, SEQ ID NO: 5).

Results

It is well known that sFlt-1 plays a major physiological role in thecornea were it a crucial anti-VEGF factor, keeping the cornea avascular,a state which is imperative for clear vision. In mammals, sFlt-1 isexpressed by the epithelia of the cornea. In order for the inventors ofthe present invention to examine whether sFlt-14 is also expressed inthe human cornea, specific sFlt-14 antibodies were used forimmunohistochemistry of human corneal sections. As clear from FIG.11A-B, sFlt-14 is highly expressed in the corneal epithelia. Thepresence of sFlt-14 in the human corneal epithelia was further validatedby sFlt-14 PCR analysis of several epithelia samples isolated from humancorneas (data not shown).

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. A method of diagnosing a pregnancy-associatedmedical condition associated with maternal or fetal stress in a subjectin need thereof, the method comprising: (a) detecting expression levelof sFlt-14 (SEQ ID NO: 2 or 4) in a biological sample of the subjectusing an agent capable of recognizing sFlt-14 (SEQ ID NO: 2 or 4) andnot sFlt-1 (SEQ ID NO: 10); and (b) diagnosing said subject as having apregnancy-associated medical condition associated with maternal or fetalstress based on the presence of increased level of said sFlt-14 (SEQ IDNO: 2 or 4) in said biological sample as compared to a sample of anormal pregnant subject.
 2. A method of diagnosing preeclampsia in asubject in need thereof, the method comprising: (a) detecting expressionlevel of sFlt-14 (SEQ ID NO: 2 or 4) in a biological sample of thesubject using an agent capable of recognizing sFlt-14 (SEQ ID NO: 2 or4) and not sFlt-1 (SEQ ID NO: 10); and (b) diagnosing said subject ashaving a preeclampsia based on the presence of increased level of saidsFlt-14 (SEQ ID NO: 2 or 4) in said biological sample as compared to asample of a normal pregnant subject.
 3. A method of diagnosingpreeclampsia in a subject in need thereof, the method comprising: (a)detecting expression level of sFlt-14 (SEQ ID NO: 2 or 4) in a serumsample or a placenta tissue sample of the subject using an antibodycapable of recognizing sFlt-14 (SEQ ID NO: 2 or 4) and not sFlt-1 (SEQID NO: 10); and (b) diagnosing said subject as having a preeclampsiabased on the presence of increased level of said sFlt-14 (SEQ ID NO: 2or 4) in said serum sample or said placenta tissue sample as compared toa sample of a normal pregnant subject.
 4. The method of claim 1, whereinsaid condition is selected from the group consisting of preeclampsia,gestational diabetes, gestational hypertension, fetal growth restriction(FGR), and fetal alcohol syndrome (FAS).
 5. The method of claim 1,wherein said agent is an antibody.
 6. The method of claim 2, whereinsaid agent is an antibody.
 7. The method of claim 1, wherein saidbiological sample is selected from the group consisting of a urinesample, a blood sample, a serum sample, a placenta biopsy, a chorionicvillus sample, and an amniotic fluid sample.
 8. The method of claim 2,wherein said biological sample is selected from the group consisting ofa urine sample, a blood sample, a serum sample, a placenta biopsy, achorionic villus sample, and an amniotic fluid sample.
 9. The method ofclaim 1, being effected in vitro or ex vivo.
 10. The method of claim 2,being effected in vitro or ex vivo.
 11. The method of claim 3, beingeffected in vitro or ex vivo.
 12. The method of claim 1, wherein saidbiological sample is of a gestation week 13 and on.
 13. The method ofclaim 2, wherein said biological sample is of a gestation week 13 andon.
 14. The method of claim 3, wherein said serum sample or saidplacenta tissue sample is of a gestation week 13 and on.